Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Study of Protein Structure
Protein Separation Methods
Experimental Methods of Electrophoresis - Two-Dimensional Electrophoresis

Just as in paper and Thin-Layer Chromatography, the two-dimensional Separation approach is applicable to PAGE. The experiment is usually carried out as follows. First, the mixture is separated by isoelectric focusing (IEF). Then, SDS-PAGE is performed in the perpendicular direction. The resolving power of this combined method is exceptionally high, making it possible to resolve mixtures containing 1,000 or more Proteins. O'Farrell, the pioneer of this method, suggested that it can resolve mixtures comprising up to 5,000 proteins. Two-dimensional electrophoretic maps (gels) serve as a kind of "fingerprint" characteristic of the source from which the sample was taken for analysis—a molecular-level "anatomy." Currently, such maps obtained for various human Tissues are being cataloged with the ultimate goal of utilizing this information for disease Diagnosis and tracking disease progression.

As an example, let us examine the results of an experiment performed by Patrick O'Farrell on the two-dimensional separation of E. coli proteins (see Fig. 4.11).

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Fig. 4.11 Radioautogram obtained after the separation of E. coli proteins grown in a medium with 14C-labeled Amino Acids

E. coli Cells were grown in a medium containing amino acids labeled with the radioactive isotope 14С. The cells were then lysed by sonication. A 25 µL aliquot of the lysate (=10 µg of protein) was loaded onto tube gels for IEF. Following IEF, the cylindrical gels were removed from the tubes and placed along the edge of a slab gel positioned between two Glass plates, where SDS Electrophoresis was carried out. Upon completion of the electrophoresis, the gels were placed against X-ray film to expose the radioactivity of the 14C-labeled proteins. Development of the film yielded the corresponding protein map of the E. coli Cell lysate (a total of 1,100 resolved spots).



Last update: 06/08/2026

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